Post on 22-Oct-2020
ORIGINAL PAPER
Agrobacterium-mediated genetic transformation of Fraxinusamericana hypocotyls
Kaitlin J. Palla • Paula M. Pijut
Received: 16 June 2014 / Accepted: 21 September 2014 / Published online: 30 September 2014
� Springer Science+Business Media Dordrecht (outside the USA) 2014
Abstract An Agrobacterium tumefaciens-mediated
genetic transformation system was successfully developed
for white ash (Fraxinus americana) using hypocotyls as the
initial explants. Hypocotyls isolated from mature embryos
germinated on Murashige and Skoog (MS) medium sup-
plemented with 22.2 lM 6-benzyladenine (BA) and0.5 lM thidiazuron (TDZ) were transformed usingA. tumefaciens strain EHA105 harboring the binary vector
pq35GR containing a fusion gene between neomycin
phosphotransferase (nptII) and gusA, as well as an
enhanced green fluorescent protein (EGFP). Explants were
transformed in a bacterial suspension with 100 lM aceto-syringone using 90 s sonication and 10 min vacuum infil-
tration. Putative transformed shoots representing seven
independent lines were selectively regenerated on MS
medium with 22.2 lM BA, 0.5 lM TDZ, 50 mg L-1
adenine sulfate, 10 % coconut water, 30 mg L-1 kana-
mycin, and 500 mg L-1 timentin. Timentin at 500 mg L-1
was optimal for controlling excess bacterial growth, and
transformed shoots were selected using 30 mg L-1 kana-
mycin. The presence of GUS (b-glucuronidase), nptII, andEGFP in transformed plants was confirmed by polymerase
chain reaction (PCR). Reverse transcription-PCR and
fluorescence microscopy confirmed the expression of
EGFP. Transgenic microshoots were rooted (80 %) on
woody plant medium supplemented with 4.9 lM indole-3-butyric acid, 2.9 lM indole-3-acetic acid, and 500 mg L-1
timentin, and subsequently acclimatized to the culture
room. This transformation protocol provides the frame-
work for future genetic modification of white ash to pro-
duce plant material resistant to the emerald ash borer.
Keywords Fraxinus � Genetic transformation �Organogenesis � Regeneration � White ash
Introduction
White ash (Fraxinus americana L.) is the most common
and economically valuable of the 16 ash species native to
North America. The wood of white ash is heavy, strong,
hard, and stiff, and it has high resistance to shock (Forest
Products Laboratory 2010). The wood is valued for the
production of tool handles, furniture, flooring, boats, and
baseball bats. Considered one of the best soil-improving
ash species because of its calcium-rich leaves and earth-
worm palatability, white ash is also utilized as shelter and
sustenance by a variety of wildlife. The vibrant fall color,
relatively fast growth, and tolerance to a wide range of soil
pH make it a favorite of the horticulture industry (Johnston
1939; Schlesinger 1990; Nesom 2001; Wallander 2008). In
2005, ash trees composed 5–29 % of all street trees in the
Midwest, replacing many of the elm trees in urban settings
since the advent of Dutch elm disease (MacFarlane and
Meyer 2005).
North American ash resources are currently being
threatened with extinction by the emerald ash borer (EAB),
K. J. Palla
Department of Forestry and Natural Resources, Hardwood Tree
Improvement and Regeneration Center (HTIRC), Purdue
University, 715 West State St., West Lafayette, IN 47907, USA
Present Address:
K. J. Palla
Oak Ridge National Laboratory, MS-6407,
P.O. Box 2008, Oak Ridge, TN 37831, USA
P. M. Pijut (&)USDA Forest Service, Northern Research Station, HTIRC, 715
West State St., West Lafayette, IN 47907, USA
e-mail: ppijut@purdue.edu
123
Plant Cell Tiss Organ Cult (2015) 120:631–641
DOI 10.1007/s11240-014-0630-1
Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)
(Herms and McCullough 2014). An aggressive wood-bor-
ing exotic, EAB has spread rapidly throughout at least 21
U.S. states and parts of Canada since being discovered in
southeastern Michigan in 2002 (Yu 1992; Emerald Ash
Borer Information Website 2014). Adult beetles feed on
tree foliage and bore holes into the bark, but the most
damage is a result of the larvae feeding on the phloem
tissue, forming galleries under the bark, and disrupting the
flow of nutrients (McCullough et al. 2008). EAB is fatal to
ash trees, and there is neither any known innate resistance
in native trees nor any means to completely eradicate the
beetle. Over 99 % tree mortality in a stand was probable
within 4 years of the first observed symptoms of infestation
(Knight et al. 2012).
Biological control and eradication efforts have made
limited impact on EAB populations or infestation rates, and
the beetle’s range is still rapidly expanding (Dobesberger
2002; Poland and McCullough 2006; Duan et al. 2012).
Chemical controls were deemed expensive and inappro-
priate for protecting ash trees in forested or riparian areas,
as well as environmentally undesirable (Poland and
McCullough 2006; Bauer and Londoño 2011; Hahn et al.
2011). The predicted cost of treating, removing, and
replacing urban forest trees affected by EAB was expected
to exceed $10 billion by 2019 (Kovacs et al. 2010).
Genetically engineering ash trees to express resistance to
the EAB, such as the Bacillus thuringiensis Cry genes, is a
desirable alternative for obtaining a cost-efficient, effec-
tive, and environmentally friendly means of controlling
EAB damage in the North American landscape. To date,
genetic transformation has been attempted for several
Fraxinus spp., however transgenic shoots have only been
reported for F. pennsylvanica and F. profunda (Roome
1992; Bates 1997; Du and Pijut 2009; Stevens and Pijut
2014). Bates (1997) obtained multiple shoots after trans-
forming white ash with Agrobacterium, but no shoots were
confirmed transgenic. Our study is the first report of suc-
cessfully regenerating transgenic white ash, and provides a
foundation for integrating EAB resistance genes into this
species.
Materials and methods
Plant materials
Mature white ash seeds purchased in 2011 from Sheffield’s
Seed Co., Inc. (Locke, NY) were stored in a sealed con-
tainer in the dark at 5 �C until used. Disinfestation, isola-tion of embryos, and adventitious shoot regeneration were
achieved using our previously described protocol (Palla
and Pijut 2011). Freshly isolated aseptic embryos were
cultured vertically in Magenta GA-7 vessels (Magenta
Corp., Chicago, IL; 25 embryos per vessel) containing
50 mL of pre-culture Murashige and Skoog (1962) (MS)
medium (M499; PhytoTechnology Laboratories, Shawnee
Mission, KS) with organics (100 mg L-1 myoinositol,
0.5 mg L-1 nicotinic acid, 0.5 mg L-1 pyridoxine HCl,
0.1 mg L-1 thiamine HCl, and 2 mg L-1 glycine), sup-
plemented with 22.2 lM 6-benzyladenine (BA), and0.5 lM thidiazuron (TDZ) for 5- to 15-d. Hypocotyls ofin vitro germinated seedlings were then isolated and cul-
tured horizontally on culture medium in order to determine
explant sensitivity to kanamycin and timentin for trans-
formation and selection procedures. All plant materials
were cultured on medium containing 30 g L-1 sucrose and
7 g L-1 Bacto agar (No. 214030; Becton–Dickinson,
USA), with the pH adjusted to 5.7 prior to autoclaving, and
all plant cultures were incubated at 24 ± 2 �C under a 16-hphotoperiod (80 lmol m-2 s-1), unless noted otherwise.
Effect of kanamycin and timentin on hypocotyl
regeneration
To determine the optimum concentration for selection of
transformed tissue and controlling excess Agrobacterium
growth (Cheng et al. 1998), hypocotyl explants from 5-d-
old in vitro grown seedlings were cultured horizontally on
co-culture MS medium with organics, 22.2 lM BA,0.5 lM TDZ, 50 mg L-1 adenine hemisulfate (AS), 10 %(v/v) coconut water (CW) (C195; PhytoTechnology Lab-
oratories, Shawnee Mission, KS) supplemented with 0, 5,
10, 15, 20, 30, 40, or 50 mg L-1 kanamycin or 0, 100, 200,
300, 400, 500, or 600 mg L-1 timentin in Petri plates
(100 9 25 mm; 50 mL medium). Antibiotics were each
dissolved in sterile deionized water and filter-sterilized
(0.22 lm) prior to being added to cooled autoclavedmedium. Three replicates with 12 hypocotyls each were
cultured for each treatment to determine the optimum
concentration of antibiotics to be used in transformation
and selection procedures. Regeneration response of callus
and shoot formation frequency were evaluated and recor-
ded after 6 weeks on initial induction medium.
Transformation vector and culture
The pq35GR vector (Fig. 1) was comprised of two inde-
pendently arranged bi-directional enhancer repeats driven
by the cauliflower mosaic virus 35S promoter (CaMV
35S). The marker genes incorporated into the vector were a
fusion of neomycin phosphotransferase (nptII) and gusA
(b-glucuronidase; GUS), and enhanced green fluorescentprotein (EGFP) (Li et al. 2004). Introduced into Agro-
bacterium tumefaciens strain EHA105, this vector was also
used for plant transformation studies with green and
632 Plant Cell Tiss Organ Cult (2015) 120:631–641
123
pumpkin ash (Du and Pijut 2009; Stevens and Pijut 2014).
Single Agrobacterium colonies were cultured in the dark for
2 days on a rotary shaker (150 rpm) at 28 �C in 20 mL liquidYEP medium (10 g L-1 yeast extract, 10 g L-1 bacto-pep-
tone, 5 g L-1 NaCl, at pH 7.0) supplemented with
20 mg L-1 rifampicin and 50 mg L-1 kanamycin. The
Agrobacterium-pq35GR suspension (OD600 = 0.4–0.6) was
centrifuged at 3,000 rpm for 15 min, and the pellet was re-
suspended in 20 mL liquid MS co-culture medium with
organics, containing 22.2 lM BA, 0.5 lM TDZ, 50 mg L-1
AS, 10 % CW, with the addition of 100 lM acetosyringone.The bacterial suspension was placed on the rotary shaker
(150 rpm) for 1 h prior to co-cultivation with hypocotyls.
Agrobacterium transformation and transgenic shoot
regeneration
Hypocotyls excised from 5- to 15-d-old germinated
embryos were placed in 20 mL liquid medium [MS med-
ium with organics, 22.2 lM BA, 0.5 lM TDZ, 50 mg L-1
AS, and 10 % CW] and sonicated for 90 s before being
transferred to the liquid Agrobacterium-pq35GR suspen-
sion and vacuum infiltrated (62.5 cm Hg) for 10 min.
Explants were then blotted dry on sterile filter paper and
cultured on semi-solid MS co-culture medium in Petri
plates (100 9 15 mm; 30 mL; 50 explants per plate) and
cultured in the dark for 2–3 days at 27 �C. Hypocotylswere then rinsed three times with liquid MS co-culture
medium to remove excess Agrobacterium, blotted dry, and
cultured horizontally on selection medium [MS medium
with organics, supplemented with 22.2 lM BA, 0.5 lMTDZ, 50 mg L-1 AS, 10 % CW, 30 mg L-1 kanamycin,
and 500 mg L-1 timentin; 15–20 explants per plate] for up
to 8 weeks. Explants regenerating shoot primordia were
transferred to selection-elongation medium [MS medium
with Gamborg et al. (1968) B5 vitamins plus 2 mg L-1
glycine (MSB5G) supplemented with 10 lM BA, 10 lMTDZ, 30 mg L-1 kanamycin, and 500 mg L-1 timentin].
Elongating, kanamycin-resistant shoots were evaluated for
the presence of marker genes after six or more subcultures
(4 week interval) on selection-elongation medium. Shoots
confirmed to contain transgenes were moved to multipli-
cation medium [MSB5G medium supplemented with
10 lM BA, 10 lM TDZ, and 500 mg L-1 timentin] toincrease shoot proliferation rates.
Effect of cytokinin concentration on shoot elongation
To maximize the number of microshoots elongating from
transformed hypocotyls, a factorial experiment was per-
formed to determine the optimal concentrations of BA and
TDZ. Explants initiating shoot buds on selection medium
(4–8 weeks) were transferred to MSB5G medium supple-
mented with 30 mg L-1 kanamycin, 500 mg L-1 timentin,
0, 10, 15, or 20 lM BA in combination with 0, 2.5, 5, or10 lM TDZ to induce shoot elongation. Magenta GA-7vessels containing 50 ml of medium were used, with one
explant per vessel; eight explants per treatment were tested.
Cultures were incubated for 6 weeks under a 16 h photo-
period, and the number of shoots elongated was recorded.
Fig. 1 pq35GR vectorcomposed of two independent
CaMV35S-driven enhancer
repeats corresponding to an
nptII and gusA fusion gene, and
an EGFP gene (Li et al. 2004)
Plant Cell Tiss Organ Cult (2015) 120:631–641 633
123
Rooting of transgenic microshoots
Elongated transgenic shoots (3–5 cm in length; with at
least two nodes) were pooled and randomly cultured on
woody plant medium (Lloyd and McCown 1981) [WPM;
L154; PhytoTechnology, Shawnee Mission, KS with
organics (100 mg L-1 myoinositol, 0.5 mg L-1 nicotinic
acid, 0.5 mg L-1 pyridoxine HCl, 1 mg L-1 thiamine HCl,
and 2 mg L-1 glycine] supplemented with 500 mg L-1
timentin, 4.9 lM indole-3-butyric acid (IBA) plus 0, 2.9,5.7, or 8.6 lM indole-3-acetic acid (IAA). Magenta GA-7vessels containing 50 mL of medium were used, with one
explant per vessel. Cultures were placed in the dark at
26 �C for 5 days, then cultured up to 12 weeks in the light.An overlay of 5 mL liquid rooting medium was added to
cultures each week after 6 weeks in light culture. Rooting
percentage, the number of roots per shoot, and root length
were recorded once roots were determined comparable to
previous experiments (Palla and Pijut 2011). Three repli-
cations with five shoots per treatment per replication were
conducted. Rooted microshoots were acclimatized to
ambient culture room conditions as described previously
(Palla and Pijut 2011).
Histochemical GUS assay
Transformed plant tissue (callus, hypocotyl, and leaf pri-
mordia) regenerating on selection medium containing
kanamycin was incubated overnight at 37 �C in a stainingsolution (0.1 M NaHPO4 buffer (pH 7.0), 0.5 mM K3-[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 10 mM EDTA, 800 mg
L-1 X-Gluc, 0.06 % (v/v) Triton X-100) following the
procedures described by Jefferson et al. (1987). Chloro-
phyll was removed from the tissue after staining using
20 % (v/v) commercial bleach solution (5.25 % sodium
hypochlorite) for 15–20 min to allow for visualization and
documentation of GUS expression.
Visualization of enhanced green fluorescent protein
The presence of GFP was detected using a fluorescence
stereomicroscope (Leica MZFLIII) equipped with a
470/40 nm excitation filter (GFP-Plant) and a 525/50 nm
barrier filter, and lit with an HBO 100 W mercury bulb.
Leaves from transgenic plants confirmed to contain the
nptII, GUS, and EGFP genes were examined along with
control (non-transgenic) leaves, and the presence or
absence of green fluorescence was compared. No
interference filter was used to block chlorophyll auto-
fluorescence, and the SpotTM imaging software was
used.
Molecular analysis of transgenic plant lines
Genomic DNA was isolated from leaves of seven inde-
pendent putative transgenic lines and from one control
(non-transformed) plant following either the protocol of the
Qiagen DNeasy Plant Mini Kit (Qiagen, USA) or the
procedures described by Lefort and Douglas (1999).
Polymerase chain reaction (PCR) was designed to specifi-
cally amplify nptII, GUS, and EGFP. Primers (forward
nptII-F 50-TGCTCCTGCCGAGAAAGTAT-30 and reversenptII-R 50-AATATCACGGGTAGCCAAGC-30) weredesigned to amplify a 364 bp PCR fragment for the nptII
gene. Primers (forward GUS-F 50-TGCTGTCGGCTTTAACCTCT-30 and reverse GUS-R 50-GGCACAGCACATCAAAGAGA-30) were designed to amplify a 332 bp PCRfragment for GUS. Primers (forward EGFP-51 50-ATGGTGAGCAAGGGCGAGGAGCTGT-30 and reverse EGFP-32 50-TTACTTGTACAGCTCGTCCATGCCG-30) weredesigned to amplify a 720 bp PCR fragment for the EGFP.
The PCR reaction (25 lL) consisted of 2.5 lL 109 PCRbuffer (Invitrogen), 1 lL 50 mM Mg2?, 1 lL 50 mMdNTP, 1 lL each of 10 lM nptII-F and nptII-R or 10 lMGUS-F and GUS-R or 10 lM EGFP-51 and EGFP-32primers, 2 lL DNA template (100 ng lL-1), 0.25 lL 5 UlL-1 Taq polymerase, and 16.25 lL sterile-deionizedwater. Plasmid DNA was used as a positive control; DNA
from a non-transformed plant and sterile-deionized water
served as negative controls. The nptII PCR reaction
included 2 min at 94 �C, followed by 35 cycles of 94 �Cfor 45 s, 56 �C for 45 s, 72 �C for 2 min, with a final10 min cycle at 72 �C. The GUS PCR reaction varied inthe number of cycles run (45) and the annealing tempera-
ture used (55 �C), but was otherwise identical. The EGFPPCR reaction included 4 min at 95 �C, followed by 40cycles of 95 �C for 30 s, 64 �C for 30 s, 72 �C for 1 min,with a final 7 min cycle at 72 �C. Amplified products of thePCR were visualized under UV light after gel electropho-
resis (1 % w/v agarose plus 0.5 lg mL-1 ethidiumbromide).
RNA isolation and reverse transcription-PCR analysis
Reverse transcription-PCR (RT-PCR) was performed on
transgenic plant lines to confirm transgene expression.
RNA was isolated from leaves (100 mg) of microshoots
confirmed by PCR to contain transgenes, and one control
(non-transformed) plant following the protocol of the
Qiagen RNeasy Plant Mini Kit (Qiagen, USA) and treated
with DNase I (Invitrogen, Carlsbad, CA, USA) to remove
genomic DNA. DNase-treated RNA (5 lg) was used as atemplate for reverse transcription to synthesize comple-
mentary DNA (cDNA) with AccuScript High Fidelity 1st
Strand cDNA Synthesis kit (Agilent Technologies, USA)
634 Plant Cell Tiss Organ Cult (2015) 120:631–641
123
according to the manufacturer’s instructions. PCR was
performed using cDNA created with Oligo (dT) primers to
amplify sequences specific to the EGFP gene. PCR was
also performed to amplify sequences specific to a house-
keeping 26S ribosomal RNA (rRNA) gene using cDNA
created with random primers. PCR to amplify the 720 bp
product for the EGFP gene was carried out using the
respective primers and thermal cycle as described previ-
ously. Primers (forward 26S-F 50-GTCCTAAGATGAGCTCAA-30 and reverse 26S-R 50-GGTAACTTTTCTGACACCTC-30) were designed to amplify a 160 bp PCRproduct for the 26S rRNA gene. The PCR reaction (25 lL)consisted of 2.5 lL 109 PCR buffer (Invitrogen), 1 lL50 mM Mg2?, 1 lL 50 mM dNTP, 1 lL each of 10 lM26S-F and 26S-R or 10 lM EGFP-51 and EGFP-32primers, 3 lL cDNA template (5 ng lL-1), 0.25 lL 5 UlL-1 Taq polymerase, and 15.25 lL sterile–deionizedwater. Plasmid DNA was used as a positive control, cDNA
from a non-transformed plant and sterile–deionized water
served as negative controls, and DNase-treated RNA that
had not been reverse transcribed served as template control
to monitor DNA contamination. The 26S PCR reaction
included 4 min at 95 �C, followed by 35 cycles of 94 �Cfor 30 s, 55 �C for 30 s, 72 �C for 1 min, with a final 7 mincycle at 72 �C. Amplified products of the PCR werevisualized under UV light after gel electrophoresis (1 %
agarose (w/v) plus 0.5 lg mL-1 ethidium bromide).Sta-tistical analysis SPSS (Software Version 20) (SPSS 2011)
was used to analyze data. An analysis of variance
(ANOVA) was performed using the General Linear Model
procedure on the individual replicate means by treatment
for percent shoot formation, number of shoots per hypo-
cotyl, percent root formation, number of roots per shoot,
root length, and number of lateral roots. When the ANOVA
indicated statistical significance, a Duncan’s comparison
test with an alpha level of 0.05 was used to distinguish the
differences between treatments.
Results and discussion
Effect of kanamycin and timentin on ash hypocotyl
regeneration
Hypocotyls were exposed to various concentrations of
kanamycin and timentin in order to determine the effect
that the antibiotics had on regeneration of white ash
adventitious shoots. Kanamycin is commonly used in the
selection of transformed cells, inhibiting the growth of cells
that do not have the nptII gene integrated, and concentra-
tion is critical for this selection. White ash callus and shoot
regeneration were significantly inhibited when kanamycin
concentrations reached 30 mg L-1 (Table 1). No shoot
primordia were able to regenerate at that concentration and
callus production was severely limited, with the majority of
hypocotyls turning chlorotic and necrotic after 6 weeks of
culture. Thus, we determined 30 mg L-1 kanamycin to be
the optimal concentration to use for subsequent transfor-
mation and selection, as it provided a high selection pres-
sure to ensure regenerating shoots would be stable
transformants instead of potential escapes. This agrees with
the previous study on white ash, where Bates (1997) noted
organogenic inhibition in seedlings cultured on kanamycin
concentrations C20 mg L-1. Other ash studies reported
kanamycin inhibited organogenesis at concentrations of
20 mg L-1 (Du and Pijut 2009; Stevens and Pijut 2014).
Roome (1992) observed that kanamycin as low as
10 mg L-1 limited green ash shoot production, and was
unable to regenerate any shoots at higher concentrations.
Timentin was used in this study to inhibit growth of
Agrobacterium after transformation. White ash hypocotyls
were not significantly inhibited by timentin, and the highest
shoot primordia regeneration (80.6 %) occurred on med-
ium containing 500 mg L-1 (Table 2). Similar high con-
centrations (300–400 mg L-1) were noted to have little
effect on hypocotyl regeneration for other ash and hard-
wood species (Gonzalez Padilla et al. 2003; Andrade et al.
2009; Du and Pijut 2009; Stevens and Pijut 2014).
Transformation and regeneration of plants
Germination of mature white ash seedlings was previously
noted to be variable, with a delay of 11- to 21-days before
being sorted to use for transformation (Bates 1997). By
Table 1 Effect of kanamycin concentration on percent callus for-mation and shoot regeneration of Fraxinus americana hypocotyls
Kanamycin
(mg L-1)
Callus formationa
(%)
Shoot formationa,b
(%)
0 100.0 ± 0.0a 83.3 ± 9.6a
5 66.7 ± 8.3b 50.0 ± 4.8b
10 47.2 ± 10.0bc 27.8 ± 7.3c
15 52.8 ± 12.1bc 25.0 ± 9.6c
20 41.7 ± 8.3c 11.1 ± 4.8 cd
30 19.4 ± 4.8d 0.0 ± 0.0d
40 0.0 ± 0.0d 0.0 ± 0.0d
50 0.0 ± 0.0d 0.0 ± 0.0d
Hypocotyls were placed on Murashige and Skoog medium supple-
mented with 22.2 lM 6-benzyladenine, 0.5 lM thidiazuron,50 mg L-1 adenine hemisulfate, and 10 % coconut water plus different
concentrations of kanamycin. Data taken after 6 weeks of culturea Mean ± standard error for 36 explants per treatment. Means in
each column followed by the same letter were not significantly dif-
ferent according to Duncan’s multiple comparison test (a = 0.05)b Mean ± standard error for hypocotyls that produced leaf primordia
Plant Cell Tiss Organ Cult (2015) 120:631–641 635
123
culturing aseptically extracted embryos on pre-culture
medium, not only were embryos ready for transformation
in 5 days, but germination was almost entirely uniform.
Embryos could be grown on this medium for up to 15 days
to allow for further hypocotyl growth. This extended pre-
culture may help the tissue better survive the transforma-
tion process (data not shown). Green ash hypocotyls were
seen to be hypersensitive to Agrobacterium, but a brief pre-
culture period prior to inoculation attenuated the response
(Du and Pijut 2009). The medium used to germinate the
embryos in this study appeared to serve the same purpose
as a pre-culture period, allowing hypocotyls to be freshly
isolated and used for transformation. Combining embryo
germination with a pre-culture step was shown to be ben-
eficial for plum (Gonzalez Padilla et al. 2003) and pumpkin
ash (Stevens and Pijut 2014).
Since Agrobacterium was shown to be attracted to the
compounds released by wounded plants (Satchel et al.
1985), the use of freshly isolated hypocotyls increased the
number of sites where inoculation could potentially occur.
Roome (1992) used abrasive celites to wound explants in
one transformation study, while freshly cut hypocotyls
proved effective for Gonzalez Padilla et al. (2003). Soni-
cation was used in this study to further increase the number
of superficial wound sites where gene transfer could take
place, and was combined with vacuum-infiltration to force
the Agrobacterium cells into further contact with the white
ash hypocotyl cells. Sonication in conjunction with vac-
uum-infiltration has proven valuable for the transformation
of many species, particularly when thicker explant tissue
such as hypocotyls have been used (Andrade et al. 2009;
Du and Pijut 2009; Subramanyam et al. 2011; Stevens and
Pijut 2014).
White ash hypocotyls were co-cultivated for 2- to 3-days
after being exposed to an Agrobacterium solution with an
OD600 = 0.4–0.6 to allow for gene transfer and integration.
Higher Agrobacterium concentrations and longer co-cul-
ture periods resulted in an excess of bacterial growth dur-
ing the selection process, regardless of timentin
concentration, resulting in necrosis of explant tissue (data
not shown). Similar bacterial concentrations (Sun et al.
2011) and co-culture times (Du and Pijut 2009; Stevens
and Pijut 2014) have been used with success. Six weeks
after culture on selection medium, 105 explants out of 537
were producing callus or shoot primordia, and were moved
to selection and elongation medium. Seven independent
transgenic lines were recovered after 8 weeks (1.3 %
transformation efficiency). Adventitious shoots that elon-
gated in the presence of kanamycin were continuously
cultured every 3 or 4 weeks on selection–elongation
medium for six or more subcultures. Shoot elongation
occurred at a slow rate while on medium containing
kanamycin, particularly for three out of the seven inde-
pendent lines, although shoots were green and normal in
appearance (Fig. 2a). The optimal concentration of BA and
TDZ for shoot elongation was examined and determined to
be 10 lM each (37.5 %; 1.3 ± 0.6 shoots per explant;Table 3). Liquid overlays of elongation medium have been
shown to increase the growth rate of transformed shoots
(Stevens and Pijut 2014), but failed to promote white ash
shoot growth, instead causing excess callus formation at
the base of microshoots. Once microshoots had grown
large enough to remove leaf material without damaging
growth, PCR was performed to confirm the presence of
transgenes, and then all transgenic shoots were cultured on
elongation medium without kanamycin (Fig. 2b). Shoot
elongation recovered to a normal rate after two to three
subcultures and were propagated in vitro for use in rooting
trials (Palla and Pijut 2011).
Rooting (80 %) of the seven transgenic white ash lines
recovered in this study occurred on WPM supplemented
with 500 mg L-1 timentin, 4.9 lM IBA, and 2.9 lM IAA(Table 4). These results follow what was observed in pre-
vious regeneration work (Palla and Pijut 2011). Kanamycin
has been known to negatively affect rooting (Gonzalez
Padilla et al. 2003), and was observed to limit shoot call-
using, root initiation, and root elongation in initial rooting
tests with white ash transformants obtained in our lab (data
not shown). All shoots used in the current rooting trial were
derived from lines PCR-confirmed to contain transgenes,
therefore kanamycin was omitted from the rooting medium
to enhance root initiation and elongation. As with shoot
elongation, root formation and development was slow for
the transformed shoots. Liquid overlays of medium were
observed to enhance rooting in green ash (Kim et al. 1998).
A liquid overlay of rooting medium (5 mL) every week
Table 2 Effect of timentin concentration on percent callus formationand shoot regeneration of Fraxinus americana hypocotyls
Timentin (mg L-1) Callus formationa (%) Shoot formationa,b (%)
0 100.0 ± 0.0a 91.7 ± 4.8a
50 100.0 ± 0.0a 50.0 ± 12.7c
100 100.0 ± 0.0a 72.2 ± 7.4abc
200 100.0 ± 0.0a 75.0 ± 4.8abc
300 100.0 ± 0.0a 75.0 ± 4.8abc
400 100.0 ± 0.0a 61.1 ± 10.0bc
500 100.0 ± 0.0a 80.6 ± 10.0ab
600 100.0 ± 0.0a 71.5 ± 3.4abc
Hypocotyls were placed on Murashige and Skoog medium supple-
mented with 22.2 lM 6-benzyladenine, 0.5 lM thidiazuron,50 mg L-1 adenine hemisulfate, and 10 % coconut water plus dif-
ferent concentrations of timentin. Data taken after 6 weeks of culturea Mean ± standard error for 36 explants per treatment. Means in
each column followed by the same letter were not significantly dif-
ferent according to Duncan’s multiple comparison test (a = 0.05)b Mean ± standard error for hypocotyls that produced leaf primordia
636 Plant Cell Tiss Organ Cult (2015) 120:631–641
123
after the initial 6 weeks on rooting medium proved bene-
ficial for elongating transgenic white ash roots, as well as
maintaining the health of the shoot during culture. All roots
that formed were firmly attached (Fig. 2c), although callus
formation was more abundant at the base of the transgenic
shoot and roots were thicker than had been observed during
previous regeneration work on white ash (Palla and Pijut
2011). Some shoots exhibited root initiation from nodal or
internodal stem portions in contact with the rooting med-
ium, rather than from the basal end of the shoot. Rooted
Fig. 2 Kanamycin-resistantadventitious Fraxinus
americana shoots. a Shootelongating on selection–
elongation medium [MSB5G
plus 10 lM BA, 10 lM TDZ,30 mg L-1 kanamycin, and
500 mg L-1 timentin], b PCR-confirmed transgenic shoot
proliferating on elongation
medium without kanamycin,
c root formation on PCR-confirmed transgenic shoot, d,e PCR-confirmed transgenicplants acclimatized to ambient
culture room conditions
Plant Cell Tiss Organ Cult (2015) 120:631–641 637
123
plantlets were acclimatized (100 %) to ambient culture
room conditions (Fig. 2d, e).
Analysis of transgenic plant lines
Transformed plant tissue on selection medium containing
kanamycin was exposed to a histochemical GUS assay 4-
to 6-weeks after initial culture. Transient GUS expression
was observed as a bright blue coloration in the tissue after
chlorophyll removal. Staining was apparent within hypo-
cotyl tissue, showing the successful transformation with
Agrobacterium into the plant tissue, as well as on regen-
erating callus and leaf primordia, visualizing the transient
expression of the GUS gene (Fig. 3a–c).
Genomic DNA extracted from the leaves of seven
putative transgenic lines was used in PCR analysis along
with a non-transformed white ash plant. PCR amplification
resulted in 332, 364, and 720 bp (Fig. 4, lanes 4–10) DNA
fragments from all seven independent lines, corresponding
to GUS, nptII, and EGFP, respectively. PCR products of
the corresponding size were found in the plasmid control
(Fig. 4, lane 1), but absent in both the non-transformed
plant (Fig. 4, lane 3) and the water control (Fig. 4, lane 2).
This confirmed the successful integration of the foreign
genes of interest into F. americana. Lines confirmed to be
transgenic were further analyzed using a stereomicroscope
with a 470/40 nm excitation filter to visualize fluorescence
Table 4 Effect of auxin concentration on in vitro root formation of transgenic Fraxinus americana microshoots
Treatment IAA ? IBA (lM) Rootinga,b (%) Mean no. rootsa,b Mean root lengtha,b (cm) Mean no. lateral rootsa,b
0.0 ? 4.9 13.3 ± 13.3b 0.5 ± 0.4b 0.8 ± 0.1b 0.2 ± 0.1b
2.9 ? 4.9 80.0 ± 11.5a 3.8 ± 0.8a 1.2 ± 0.3a 0.8 ± 0.3a
5.7 ? 4.9 66.7 ± 13.3a 4.1 ± 1.3a 1.0 ± 0.2a 0.6 ± 0.2ab
8.6 ? 4.9 53.3 ± 6.7a 3.1 ± 1.1ab 0.7 ± 0.2ab 0.2 ± 0.1b
Microshoots were placed on woody plant medium supplemented with 500 mg L-1 timentin, 4.9 lM indole-3-butyric acid (IBA), and variousconcentrations of indole-3- acetic acid (IAA). An overlay of liquid medium was added every week after 6 weeks of culture in the light. Data
taken after 5 day dark culture followed by 8–12 weeks light culturea Values represent the means ± standard errors for 15 explants per treatmentb Means in each column followed by the same letter were not significantly different according to Duncan’s multiple comparison test (a = 0.05)
Fig. 3 Transient GUS expression in Fraxinus americana. Expression in a hypocotyl tissue, b callus, and c leaf primordia
Table 3 Effect of cytokinin concentration on elongation of trans-genic Fraxinus americana microshoots
BA (lM) TDZ (lM) Shoot elongationa,b (%) Mean no. shoota,b
0.0 0.0 0.0 ± 0.0b 0.0 ± 0.0b
10.0 0.0 0.0 ± 0.0b 0.0 ± 0.0b
15.0 0.0 12.5 ± 70.7ab 2.0 ± 0.0a
20.0 0.0 12.5 ± 35.4ab 1.0 ± 0.0ab
0.0 2.5 0.0 ± 0.0b 0.0 ± 0.0b
10.0 2.5 0.0 ± 0.0b 0.0 ± 0.0b
15.0 2.5 12.5 ± 35.4ab 1.0 ± 0.0ab
20.0 2.5 0.0 ± 0.0b 0.0 ± 0.0b
0.0 5.0 0.0 ± 0.0b 0.0 ± 0.0b
10.0 5.0 0.0 ± 0.0 b 0.0 ± 0.0b
15.0 5.0 12.5 ± 70.7ab 2.0 ± 0.0a
20.0 5.0 0.0 ± 0.0b 0.0 ± 0.0b
0.0 10.0 0.0 ± 0.0b 0.0 ± 0.0b
10.0 10.0 37.5 ± 75.6a 1.3 ± 0.6ab
15.0 10.0 0.0 ± 0.0b 0.0 ± 0.0b
20.0 10.0 12.5 ± 35.4ab 1.0 ± 0.0ab
Hypocotyls forming shoot primordia were placed on Murashige and Skoog
medium with Gamborg et al. (1968) B5 vitamins plus 2 mg L-1 glycine
supplemented with 30 mg L-1 kanamycin, 500 mg L-1 timentin, and
various concentrations of 6-benzyladenine (BA) and thidiazuron (TDZ).
Data taken after 6 weeks of culturea Values represent the means ± standard errors for eight explants per
treatmentb Means in each column followed by the same letter were not significantly
different according to Duncan’s multiple comparison test (a = 0.05)
638 Plant Cell Tiss Organ Cult (2015) 120:631–641
123
associated with the presence of the EGFP gene. Young
leaves from PCR-confirmed shoots were compared in vivo
against non-transformed control leaves. Bright green por-
tions of fluorescence were apparent only on leaves from
transgenic shoots (Fig. 5), confirming both the presence
and the expression of the EGFP gene.
The expression of the EGFP gene in leaves of transgenic
plants was also determined by RT-PCR analysis. The
amplification of the 720 bp fragment of EGFP was found in
all seven transgenic plant lines (Fig. 6, lanes 5–11). A
corresponding band was found in the plasmid control
(Fig. 6, lane 4), but not in the water control (Fig. 6, lane 1),
the RNA control (Fig. 6, lane 2), or the non-transformed
plant (Fig. 6, lane 3). This confirmed the expression of the
EGFP gene in all seven transformed lines, as well as the
success of the reverse transcription of RNA into cDNA and
the removal of DNA contamination. Yang et al. (2006)
noted that Agrobacterium cells can potentially survive
selection, persisting around the cells in leaves, stems, and
roots of transgenic plants for 24 months while still in the
presence of kanamycin. The highly conserved region of the
housekeeping 26S rRNA found in plants was used as a
control to validate RT-PCR results, as was used to evaluate
the stable transformation of Cornus (Liu et al. 2013). All
seven transgenic lines (Fig. 7, lanes 5–11) as well as the
non-transformed plant (Fig. 7, lane 4) showed amplifica-
tion of the 26S rRNA fragment, confirming that the gene
Fig. 4 PCR analysis of genomic DNA isolated from leaves of non-transformed and transgenic white ash for amplification of 332-bp
GUS, 364-bp nptII gene, and 720-bp EGFP, respectively. M 100 bp
molecular marker, lane 1 positive control (pq35GR), lane 2 water
control, lane 3 negative control (non-transformed plant), lanes 4–10
(putative transgenic shoots), M 100 bp molecular marker
Fig. 5 EGFP visualization oftransformed leaf. a Transformedleaf from microshoot confirmed
to harbor GUS, nptII, and EGFP
and b control non-transformedleaf under white light. Same
c transformed leaf and d non-transformed leaf as seen through
EGFP excitation filter (470/
40 nm)
Fig. 6 RT-PCR analysis of cDNA isolated from leaves of non-transformed and transgenic white ash for amplification of 720-bp
EGFP. M 100 bp molecular marker, lane 1 water control, lane 2
DNase-treated RNA template control, lane 3 non-transformed plant
(negative control), lane 4 pq35GR plasmid DNA (positive control),
lanes 5–11 transgenic shoots, M 100 bp molecular marker
Plant Cell Tiss Organ Cult (2015) 120:631–641 639
123
expression detected during RT-PCR was the result of an
integrated transgene rather than Agrobacterium contami-
nation. Contamination of the PCR reaction was apparent
however, resulting in faint banding for the water control
(Fig. 7, lane 2) and plasmid control (Fig. 7, lane 1). Several
attempts were made to eliminate the source of this con-
tamination and obtain a clearer image.
Conclusions
Transgenic white ash plants were successfully developed
using an Agrobacterium–mediated transformation and
regeneration protocol for hypocotyl explants. Hypocotyls
germinated up to 15 days on pre-culture medium contain-
ing cytokinins were exposed to sonication and vacuum-
infiltrated with Agrobacterium strain EHA105 harboring
the pq35GR vector. Selection of transformed cells was
optimal with 30 mg L-1 kanamycin and excess Agrobac-
terium growth was controlled with 500 mg L-1 timentin
without limiting organogenic potential. The integration of
marker genes was confirmed through PCR analysis, GUS
staining, GFP visualization, and RT-PCR analysis. Optimal
rooting of transgenic shoots (80 %) occurred on WPM with
500 mg L-1 timentin, 4.9 lM IBA, plus 2.9 lM IAA. Thisis the first report of successfully regenerating transgenic
white ash plants. Similar methods have been reported
successful for green ash and pumpkin ash (Du and Pijut
2009; Stevens and Pijut 2014). This protocol provides a
method for future development of EAB-resistant white ash
trees. Studies are currently underway to integrate the
Bacillus thuringiensis Cry8Da toxin protein into F. amer-
icana, with the goal of imparting resistance to the beetle.
Acknowledgments The authors gratefully acknowledge Drs. Zong-Ming (Max) Cheng, Manjul Dutt, and Vibha Srivastava for their
constructive review and suggestions for the improvement of this
manuscript. The authors would also like to acknowledge Dr. Jody
Banks (Purdue University) for use of the fluorescence stereomicro-
scope and Dr. Dennis J. Gray (University of Florida) for the trans-
formation vector pq35GR. This research was supported by a Fred M.
van Eck scholarship for Purdue University to Kaitlin J. Palla, with
partial funding from the U.S. Department of Agriculture-Animal and
Plant Health Inspection Service-Plant Protection and Quarantine-
Center for Plant Health Science and Technology. Mention of a
trademark, proprietary product, or vendor does not constitute a
guarantee or warranty of the product by the U.S. Department of
Agriculture and does not imply its approval to the exclusion of other
products or vendors that also may be suitable.
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http://www.na.fs.fed.ushttp://www.na.fs.fed.ushttps://plants.usda.gov/plantguide/pdf/cs_fram2.pdfhttps://plants.usda.gov/plantguide/pdf/cs_fram2.pdfhttp://dx.doi.org/10.1007/s00299-014-1562-2http://dx.doi.org/10.1007/s00299-014-1562-2
Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotylsAbstractIntroductionMaterials and methodsPlant materialsEffect of kanamycin and timentin on hypocotyl regenerationTransformation vector and cultureAgrobacterium transformation and transgenic shoot regenerationEffect of cytokinin concentration on shoot elongationRooting of transgenic microshootsHistochemical GUS assayVisualization of enhanced green fluorescent proteinMolecular analysis of transgenic plant linesRNA isolation and reverse transcription-PCR analysis
Results and discussionEffect of kanamycin and timentin on ash hypocotyl regenerationTransformation and regeneration of plantsAnalysis of transgenic plant lines
ConclusionsAcknowledgmentsReferences